期刊
MICROORGANISMS
卷 9, 期 3, 页码 -出版社
MDPI
DOI: 10.3390/microorganisms9030472
关键词
L-alanine; starvation; stress-induced mutagenesis; auxotrophic mutant; prototrophic mutant; Escherichia coli
类别
资金
- Japan Society for the Promotion of Science, JSPS KAKENHI grant [JP24658067]
- Core-to-Core Program (Advanced Research Networks) of the Japan Society for the Promotion of Science
In E. coli, stress-induced mutagenesis led to the emergence of L-alanine prototrophic mutants when grown in L-alanine-deficient medium, with the mutants carrying point mutations in key metabolic pathways. Addition of pyruvate relieved the starvation stress and prevented the emergence of L-alanine prototrophic mutants, highlighting the role of pyruvate in inducing L-alanine prototrophy.
In Escherichia coli, L-alanine is synthesized by three isozymes: YfbQ, YfdZ, and AvtA. When an E. coli L-alanine auxotrophic isogenic mutant lacking the three isozymes was grown on L-alanine-deficient minimal agar medium, L-alanine prototrophic mutants emerged considerably more frequently than by spontaneous mutation; the emergence frequency increased over time, and, in an L-alanine-supplemented minimal medium, correlated inversely with L-alanine concentration, indicating that the mutants were derived through stress-induced mutagenesis. Whole-genome analysis of 40 independent L-alanine prototrophic mutants identified 16 and 18 clones harboring point mutation(s) in pyruvate dehydrogenase complex and phosphotransacetylase-acetate kinase pathway, which respectively produce acetyl coenzyme A and acetate from pyruvate. When two point mutations identified in L-alanine prototrophic mutants, in pta (D656A) and aceE (G147D), were individually introduced into the original L-alanine auxotroph, the isogenic mutants exhibited almost identical growth recovery as the respective cognate mutants. Each original- and isogenic-clone pair carrying the pta or aceE mutation showed extremely low phosphotransacetylase or pyruvate dehydrogenase activity, respectively. Lastly, extracellularly-added pyruvate, which dose-dependently supported L-alanine auxotroph growth, relieved the L-alanine starvation stress, preventing the emergence of L-alanine prototrophic mutants. Thus, L-alanine starvation-provoked stress-induced mutagenesis in the L-alanine auxotroph could lead to intracellular pyruvate increase, which eventually induces L-alanine prototrophy.
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